In many applications it is desirable to enrich, or alternatively deplete, certain cell populations in a biological sample. The fields of hematology, immunology and oncology rely on samples of peripheral blood and cell suspensions from related tissues such as bone marrow, spleen, thymus and fetal liver. The separation of specific cell types from these heterogeneous samples is key to research in these fields. Purified populations of immune cells such as T cells and B cells are necessary for the study of immune function and are used in immunotherapy. Investigation of the cellular, molecular and biochemical processes require analysis of certain cell types in isolation. Numerous techniques have been used to isolate lymphocyte subsets such as T cells, B cells and natural killer (NK) cells and granulocytes such as neutrophils, basophils and eosinophils.
Hematopoietic cells and immune cells have been separated on the basis of physical characteristics such as density and through direct targeting with magnetic particles. The advent of monoclonal antibodies against cell surface antigens has greatly expanded the potential to distinguish and separate distinct cell types. There are two basic approaches to separating cell populations from blood and related cell suspensions using monoclonal antibodies. They differ in whether it is the desired or undesired cells which are distinguished/labelled with the antibody(s). In positive selection techniques, the desired cells are labelled with antibodies and removed from the remaining unlabelled/unwanted cells. In negative selection, the unwanted cells are labelled and removed. Antibody and complement treatment and the use of immunotoxins is a negative selection technique, whereas fluorescence assisted cell sorting (FACS) and most bulk immunoadsorption techniques can be adapted to both positive and negative selection. In immunoadsorption techniques, cells are selected with monoclonal antibodies and preferentially bound to a surface which can be removed from the remainder of the cells e.g. column of beads, flasks, magnetic particles. Immunoadsorption techniques have won favour clinically and in research because they maintain the high specificity of targeting cells with monoclonal antibodies, but unlike FACS, they can be scaled up to deal directly with the large numbers of cells in a clinical harvest and they avoid the dangers of using cytotoxic reagents such as immunotoxins and complement. They do however, require the use of a “device” or cell separation surface such as a column of beads, panning flask or magnet.
Density separations are commonly used to isolate peripheral blood mononuclear cells from granulocytes and erythrocytes. FICOLL™ (GE Healthcare Life Sciences, Chalfont, United Kingdom) is the most popular density solution used for this application. In a FICOLL™ density separation whole blood is layered over FICOLL™, and then centrifuged. The erythrocytes and granulocytes settle with the pellet and the mononuclear cells remain at the FICOLL:plasma interface. The success of this technique relies on the difference in density between mononuclear cells and granulocytes. If whole blood is stored for more than 24 hours the granulocytes change density and will not pellet with the erythrocytes. Suspensions of pure mononuclear cells cannot be obtained from stored blood or samples with altered cell density in a single density separation. RosetteSep is a commercially available immunodensity cell separation product from STEMCELL Technologies Inc. (Vancouver, Canada) that utilizes density gradient centrifugation to enrich cells from erythrocyte containing samples such as human peripheral whole blood by forming immunorosettes between undesired nucleated cells with erythrocytes. Although immunodensity cell separation can efficiently enrich nucleated cell populations from human peripheral whole blood, there can be residual erythrocyte contamination in the final enriched cell sample. For more information on the use of density gradient centrifugation for the separation of immunorosettes see U.S. Pat. No. 6,448,075 to Thomas, which is incorporated herein by reference.
Current cell separation methods using immunomagnetic negative selection methods for the isolation of immune cells, hematopoietic stem cells and circulating epithelial tumor cells typically involve an initial step to deplete erythrocytes followed by antibody mediated adherence to a device or artificial particle. Several commercial cell separation products are available that utilize a pre-processing step to reduce the erythrocyte content of peripheral whole blood such as hypotonic erythrocyte lysis, FICOLL™ density centrifugation or a gravity sedimentation approach prior immunomagnetic cell separation (Miltenyi Biotec Inc., Gladbach, Germany, Life Technologies Corp., Carlsbad, USA., STEMCELL Technologies Inc., Vancouver, Canada). Recently, a new method has been described (see PCT/EP2012/073083, which is incorporated herein by reference) that combines gravity sedimentation of erythrocytes with immunomagnetic cell separation in a single step to enrich for desired cells from peripheral human whole blood. This method however requires an additional immunomagnetic depletion step to further reduce erythrocyte contamination in the enriched sample.
In view of the foregoing, there is a need in the art to provide novel methods for improving erythrocyte removal and separating desired cells or removing unwanted cells from biological samples containing erythrocytes without the need for pre-processing or using time consuming gravity sedimentation approaches. There is currently no soley immunomagnetic cell separation method for the negative selection of cells directly from erythrocyte containing samples such as, but not limited to human peripheral whole blood without reducing the erythrocyte content by density centrifugation, agglutination or hypotonic lysis of whole blood either prior to or in combination with immunomagnetic cell separation.